Methods and compositions using molecular decoyants for ameliorating the undesired effects of foreign agents which bind to endogenous receptors

ABSTRACT

Molecular decoyants are chemical structures which functionally resemble an endogenous receptor. They may be used for the treatment of humans or other animals which have been subjected to a foreign agent which exerts an undesired effect only after first binding to that endogenous receptor. A decoyant is a fraction of an endogenous receptor which retains the essential elements of the binding site of the receptor for the foreign agent or is a synthetic or biosynthetic derivative thereof. The α184-200 amino acid sequence of Torpedo californica acetylcholine receptor is an example of a decoyant which protects against curarimimetic neurotoxins. Another example is the fraction of the CD4 receptor which retains the essential elements of the receptor for protection against HIV.

FIELD OF THE INVENTION

The present invention relates to substances useful for the treatment ofanimals, including humans, which have been subjected to a foreign agent,which agent exerts an undesired effect only after first binding to anendogenous receptor. More particularly, the present invention relates tosuch substances, and methods of use thereof, which are directly orindirectly derived from the natural endogenous receptor.

BACKGROUND OF THE INVENTION

In order to exert their action in a living body, many pathogenic andtoxic agents, such as viruses, bacteria and also toxins and poisons,become attached to specific binding sites, e.g., to cell surfacereceptors. Such binding phenomena may be necessary as a first step inviral infectivity or may be the essence of toxic inactivation of acritical physiological function. A receptor is a cellular component thatinteracts with a specific ligand. Ligands classified as agonists, whenbound to their receptors, activate an effector system and trigger abioresponse. Ligands classified as antagonists depress receptors orinhibit the action of the agonist. When, for example, cobra venom orcurare is attached to a cholinergic receptor, the binding ofacetylcholine is prevented. Prevention of such formation of certainligand-receptor complexes should be beneficial in combatting thedeleterious effects of the pathogenic or toxic agent.

Prevention of complex formation can be achieved via a number ofbasically different approaches. For example, the generation ofanti-toxin antisera has proven to be a useful approach in the treatmentof snake bites. Immunological inactivation of viruses and bacteria isthe essence of vaccination. In both of these cases the infiltratingsubstance is intercepted by highly stereospecific immunoglobulins whichin turn prevent the substance from reaching its target of action.

Alternatively, a different approach has recently been suggestedaccording to which an analogue of the foreign material is used topreoccupy the host receptor binding site, and thus viruses or bacteriaare prevented from associating with the tissue they normally wouldinfect.

These known approaches suffer certain basic drawbacks. Immunologicalinactivation is "ligand-specific". Furthermore, many bacteria andviruses have the capability of periodically modifying their immunogenicepitopes by random mutations and recombination processes, therebyrendering the immunoglobulin ineffective. The use of ligand analogues is"receptor-specific". However, by definition, such analogues occupy thereceptor preventing its functionability.

The present invention employs a novel approach to the problem ofprevention of formation of such ligand-receptor complexes which approachis "receptor-specific" yet does not prevent the functionability of thenative receptor.

SUMMARY OF THE INVENTION

It is an object of the present invention to obviate the drawbacks of theprior art.

It is a further object of the present invention to provide a novelapproach for the prevention of formation of ligand-receptor complexeswhich approach is "receptor-specific" yet does not prevent thefunctionability of the receptor sites.

It is another object of the present invention to provide moleculardecoyants which are not substantially larger than the molecularstructure of the ligand binding site in the native receptor and whichcan be used to bind pathogenic or toxic agents in vivo in a"target-specific" manner.

It is yet another object of the present invention to provide substanceswhich are small enough as to be substantially non-immunogenic and whichact as decoyants in vivo to compete with the natural binding sites andintercept the specific ligands and inactivate them.

It is still a further object of the present invention to provide amethod for the treatment of animals, including humans, which have beensubjected to foreign agents which exert an undesired effect only afterfirst binding to an endogenous receptor.

It is still another object of the present invention to provide sucha-method by administering a molecular decoyant which mimics thefunctional attributes of the binding site of the endogenous receptor.

These and other objects of the present invention will be furtherunderstood after consideration of the following detailed description ofpreferred embodiments in conjunction with the attached drawings.

The novel solution to the problem of prevention of formation ofligand-receptor complexes, when the ligand is a foreign agent whichexerts an undesired effect by specifically associating with anendogenous receptor, involves the identification of the molecularstructure of the ligand binding site in the native receptor and theproduction of mimic ligand binding sites. These sites can be used invivo to bind toxins or viruses or any other foreign agent in a"target-specific manner". Thus, the mimic ligand binding sites of thepresent invention compete in the animal's body with natural bindingsites, thus acting as decoys. Such substances have been denominated"molecular decoyants" by the present inventor.

It is believed that natural receptors are rather large structures,comprising some hundreds of amino acids, and can be as large as amolecular weight of about 250,000. The specific binding site, however,is much smaller and this opens up the possibility of preparingartificial, synthetic binding sites, which are effective in bindingspecific viruses, bacteria, toxins, etc., yet which comprise a muchsmaller number of amino acids, preferably less than 100, and which havetherefore a considerably lower molecular weight and thus acorrespondingly lower immunogenicity. It has been found possible toprepare such binding-site-mimicking molecular decoyants which areadapted to bind specific ligands with a size on the order of about 20amino acids. Such rather small peptide structures can be prepared byphysically dividing the endogenous receptor or they can be preparedsynthetically by the preparative procedures of peptide chemistry, suchas Merrifield synthesis, or by genetic engineering. This opens up thepossibility of large scale production of such specific polypeptidestructures and their use as active materials in the treatment of animalssubjected to pathogenic or toxic agents.

Since molecular decoyants bind to the ligand at the very site requiredby the ligand to bind with the endogenous receptor required to exert itsundesired effect, this site cannot be changed by the ligand withoutdeactivating itself. Thus, decoyants are much more reliable thanimmunoglobulins and have a long term effect.

The present invention is of a very wide applicability as moleculardecoyants specific to a wide variety of ligands can be produced. Thepresent invention includes prophylactic as well as therapeuticcompositions which contain the active molecular decoyant structures inan adequate concentration and quantity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the process steps for obtaining the 17 amino acid sequenceWKHWVYYTCCPDTPYLD by recombinant DNA technology.

FIG. 2 shows the results of the separation on polyacrylamide gels ofvarious samples of an R4137 clone cultured for induction of the trpEfusion protein. The cells were either solubilized in sample buffer (T)or sonicated in high salt buffer (500 mM) and centrifuged. Thesupernatant (S₁) contained 40-60% of the fusion protein as did thepellet (Pi). The pellet was further extracted with water to generate asupernatant (S₂) which contained some 15% of the original fusion proteincontent and a pellet (P₂). After polyacrylamide gel electrophoresis(PAGE), the samples were either stained with Coomassie brilliant blue(top) or blotted and overlayed with ¹²⁵ I-labeled α-bungarotoxin (BTX)followed by autoradiography (bottom). The arrowheads indicate theposition of the fusion protein. Numbers indicate relative molecular massin kDa.

FIG. 3 is a graph showing the concentration of bound toxin at specifictimes (C_(t)) divided by that reached at equilibrium (C_(eq)) afterincubation of R4137 with ¹²⁵ I-labeled BTX for different periods of timeas indicated. The concentrations were measured after applying aliquotsto positively charged membrane filter discs. The time was measured inminutes in panel A or seconds in panel B.

FIG. 4 is a graph showing the Scatchard analysis of toxin binding toR4137. After incubation with different concentrations of ¹²⁵ I-labeledBTX until equilibrium was achieved (30 min), net bound BTX wasdetermined by adding a 1,000 fold excess of nonradioactive BTX and thebound versus the free toxin for each point was calculated.

FIG. 5 is a graph showing competition of BTX binding. The percent of ¹²⁵I-labeled BTX (2·10⁻⁸ M) is plotted after mixing with ever increasingconcentrations of: non-labeled BTX (), cobratoxin (Δ), decamethonium(⋄), d-tubocurarine (◯), NaCl (□), carbamylcholine (x) or glycine ().The mixtures were incubated with equal amounts of R4137 for 30 min at25° C. and the net amount of bound radioactive toxin was determined.

FIG. 6A is a graph plotting the amount of bound ¹²⁵ I-labeled BTX as afunction of the total amount of ¹²⁵ I-labeled BTX applied to aconcanavalin-A column having AcChoR immobilized thereon.

FIG. 6B is a graph plotting the amount of bound ¹²⁵ I-labeled BTX on aconcanavalin-A column having AcChoR immobilized thereon as a function ofthe amount of R4137 applied to the column after different amounts ofR4137 and constant amounts of ¹²⁵ I-labeled BTX were applied thereto.

FIG. 7 is a graph showing the effect of R4137 on the survival rate ofd-tubocurarine injected mice. Two groups of Balb/C mice (35 in each)were injected with either pATH2 or R4137 (approx. 3 nmole BTX bindingsites/mouse) intraperitoneally. Five minutes later the mice were givend-tubocurarine (approx. 15 nmole, 9 μg/mouse, subcutaneously). Thenumber of survivors as a function of time after the injection of toxinis shown (the data are derived from experiments 2 and 3 of Table 1).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

While the present invention is applicable to molecular decoyants whichmimic the binding site of any endogenous receptor, it will be discussedin detail with respect to the cholinergic binding site. It is known thatα-bungarotoxin (BTX), which is a snake venom α-neurotoxin, exerts itstoxic effect by blocking the binding of cholinergic ligands to thenicotinic acetylcholine receptor (AcChoR).

The neuro-muscular junction is the site where nerves meet with musclefibers. The point of contact is the synapse and is characterized by thefact that the nerve and muscle are not actually physically connected,but rather form a chemical junction. When the nerve pulse reaches thetip of the axon, acetylcholine, the neurotransmitter, is secreted intothe gap between the nerve and muscle, i.e., "the synaptic cleft". Theacetylcholine is bound by its receptor which is situated on theextracellular membrane of the muscle, the post-synaptic side of thejunction. The binding of two molecules of acetylcholine to theirreceptor causes an ion-channel to open, the membrane to depolarize andeventually leads to muscle contraction.

BTX is an antagonist which binds to AcChoR, thereby preventingacetylcholine from reaching its receptor and preventing musclecontraction. To make a molecular decoyant for the treatment of animalssubjected to BTX, one must first identify the particular BTX bindingsite. The preferred method for doing this is by means of ligand overlayof protein blots. Once the binding site is identified, the minimalsequence may be produced. Upon the administration of such a sequence,the decoy will mimic the binding site and bind with BTX, therebyblocking the undesired activity of the toxin.

The specific binding site for BTX on the AcChoR is known to be situatedon the α-subunit thereof. The minimal essential elements of the bindingsite which will still permit selective and specific binding ofreasonable affinity to BTX may be further identified by means of proteinblotting. The techniques of protein blotting are discussed in detail inGershoni, "Protein Blotting: A Manual" in Methods of BiochemicalAnalysis, edited by David Glick, John Wiley and Sons, vol. 33, pp. 1-55,1988, the entire contents of which are hereby incorporated by reference.The process involves transferring resolved polypeptides fromchromatographic gels to immobilizing matrices.

BTX is a polypeptide toxin (74 amino acid residues) which can beiodinated and binds the receptor with an affinity of K_(D) =10⁻¹¹ M.Purified AcChoR is subjected to polyacrylamide gel electrophoresis(PAGE) under mild denaturation without boiling of the sample and use oflithium dodecyl sulfate, instead of sodium dodecyl sulfate (SDS). Blotsare then prepared and probed with ¹²⁵ I-labelled BTX. Such experimentsshow that the α-subunit of the AcChoR is labelled (Gershoni et al,"Binding of α-Bungarotoxin to Isolated α-Subunit of the AcetylcholineReceptor of Tornedo californica: Quantitative Analysis with ProteinBlots", Proc. Natl. Acad. Sci. (USA), 80: 493-4977 (1983)). Theα-subunit is then proteolysed and then protein blots thereof probed withalkaline-phosphatase hydrazide, concanavalin-A, BTX and sequencespecific antibodies which together have allowed the mapping of the toxicbinding site to the region α-160-330 and more particularly α-160-210 andeven more specifically to the region α-180-200 (see Neumann et al,"Mapping of the α-Bungarotoxin Binding Site with the α-Subunit of theAcetylcholine Receptor", Proc. Natl, Acad. Sci. (USA), 83:3008-3011(1986), the entire contents of which are hereby incorporated byreference).

In order to determine the cholinergic binding site more particularly, anumber of synthetic peptides were prepared and their capacity to bindcholinergic ligands was assessed. Of the many peptides tested the onlyones that were capable of BTX binding included the sequence α-185-196.This peptide was found to have low affinity (10⁻⁵ M) yet highly specificBTX binding ability. Higher affinity (10⁻⁷ M) was found for the sequenceα-173-204.

BTX-binding sequences can also be produced by recombinant DNAtechniques, sub-clones of cDNA of α-sub-units of mouse or Torpedocalifornica were prepared using expression vectors. The trpE fusionvector pATH2 was used. Restriction fragments of the plasmid p42, a cDNAclone of the α-subunit of Torpedo californica AcChoR, were purified on1% agarose gels. Preparative quantities of plasmids were obtained, andligations in transformations of E. coli strain HB101 were performed bythe methods of Maniatis et al., "Molecular Cloning: A LaboratoryManual", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982.By such means fusion proteins were prepared in E. coli transformants(Gershoni, "Expression of the α-Bungarotoxin Binding Site of theNicotinic Acetylcholine Receptor by Escherichia coli Transformants",Proc. Natl. Acad. sci. (USA), 84, 4318-4321 (1987), the entire contentsof which are hereby incorporated by reference). These fusion proteinswere shown to specifically bind BTX (affinity: 10⁻⁷ M). Thus, forexample, bacterially expressed proteins containing α-166-200 bind toxinwhereas those expressing α-201-315 do not.

Two oligonucleotides were prepared as described in FIG. 1(1). They weredesigned using bacterially preferred codons to code for the amino acidsequence:Gly-Ile-Glu-Gly-Arg-Trp-Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr-Pro-Tyr-Leu-Asp,which includes α184-200 of Torpedo californica AcChoR as well as apentapeptide introduced N-terminal to residue W184. The first glycine isthe result of the trinucleotide dGGG which is necessary for themaintenance of a functional SmaI site. The following sequence IEGR isthe specific cleavage site for the coagulation factor Xa (CFX). Thus theexpressed fusion protein becomes cleavable with this enzyme therebyenabling the eventual release of the site of interest, i.e., the 17amino acid sequence: WKHWVYYTCCPDTPYLD. The two oligonucleotides weremixed at a ratio of 1:1 heated together and allowed to anneal forming a9-base pair duplex. The complementary strands were then enzymatically"filled in" using Klenow polymerase.(1μl; 5 units), as shown in FIG.1(2), after which the DNA was phenol-extracted and precipitated inethanol using glycogen as a carrier. The construct was then ligated withSmaI-cut, purified pATH2 expression vector. The ligated vectors wereused to transform E. coli strain HB101, all in accordance with thetechniques described in Aronheim et al, "Characterization of the Bindingof α-Bungarotoxin to Bacterially-Expressed Cholinergic Binding Sites",J. Biol. Chem., 263, 20:9933-9937 (1988), the entire contents of whichare hereby incorporated by reference.

The transformed bacterial clones which contained the insert in properorientation were selected by, ¹²⁵ I-labeled BTX overlay of colony-blots.These transformants were found to produce an efficient toxin-bindingfusion-protein (36kDa designated R4137, FIG. 2). R4137 could be highlyenriched by sonicating the transformed cells in phosphate buffer+500 mMsalt. Centrifugation resulted in a pellet which contained 40-60% of thetotal R4137 and little more (FIG. 2, P₁). This pellet could be extractedwith water to produce a soluble fraction which was predominantly R4137(FIG. 2, S₂).

The R4137 in the S₂ fraction was biochemically characterized bymeasurement of toxin binding thereto in accordance with the techniquesdescribed in Gershoni et al, "Molecular Decoys: Ligand-BindingRecombinant Proteins Protect Mice from Curarimimetic Neurotoxins", Proc.Natl. Acad. Sci (USA), 85, 4087-4089 (1988), the entire contents ofwhich are hereby incorporated by reference. In essence, aliquots of S₂were incubated with ¹²⁵ I-labelled BTX. Then the mixture was filteredthrough a charge modified membrane filter to separate the bound versusfree toxin. The filters were then counted for radioactivity. Using thistechnique it was found that BTX associates with R4137 in a pseudo firstorder kinetic which reaches 50% completion within 40 sec and about 90%completion after 6 min (FIG. 3). Measurements of toxin binding atequilibrium (30 min reaction) showed that toxin bound to R4137 with aK_(D=) 1.2×10⁻⁷ M (FIG. 4). This binding could be competed with othercholinergic ligands with a progression of efficiencies which is similarto that known for the intact receptor (FIG. 5).

Accordingly, R4137 demonstrates that the 17 amino acid sequence:α-184-Trp-Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr-Pro-Tyr-Leu-Asp-200is sufficient for BTX binding. Although this binding is appreciably lessthan that of the intact receptor, it does resemble the bindingcharacteristics of the complete α-subunit (437 amino acids) of AcChoR.

In order to establish whether R4137 could be used as a treatment foranimals which have been subjected to a neurotoxin, an experiment wasconducted to establish that R4137 can compete against intact AcChoR fora common and limiting pool of BTX. In order to do this, one must notonly separate bound BTX from free, but one must also distinguish betweenAcChoR-bound versus R4137-bound toxin. In order to do so, AcChoR wasfirst bound to a concanavalin-A column. This was found to have little orno effect on toxin binding (FIG. 6A). Then such immobilized AcChoR wasmixed with different concentrations of R4137 and constant amounts of ¹²⁵I-labelled-BTX. Afterwards, the columns were washed by repeatedcentrifugation/resuspension and the amount of radioactivity associatedwith the column was determined. As can be seen in (FIG. 6B), R4137effectively competes for BTX against AcChoR.

Finally, R4137 was tested for competition with AcChoR in vivo. Male andfemale mice (approximately 5 weeks, 20-25g), both inbred (Balb/C) andoutbred (CD1) strains, were first injected intraperitoneally with R4137or a placebo-a similar fraction derived from bacteria transformed withthe unmodified vector pATH2 (these cells have no toxin-bindingcapacity). Five minutes later all the mice were challenged with variousamount of d-tubocurarine or α-cobratoxin (CTX). The dose was gauged tocause 80% death of untreated mice. The toxins were administered bysubcutaneous injection to the nape of the necks of the animals. Theanimals were observed and the number of deaths during the course of twohours after the toxin injection was recorded. The results are shown inTable 1. As shown in FIG. 7 the survival rate of the R4137 treated miceis markedly improved over the control and the absolute number ofrecoveries is at least 300% better than those that were given theplacebo.

                  TABLE 1                                                         ______________________________________                                             Bacterial                                                                     protein               Toxin                                                                              Dose  No. of                                  Exp. (i.p.)   Strain  Sex  (s.c.)                                                                             mg/kg mice  Survivors                         ______________________________________                                        1    pATH2    Balb/C  M    Tubo 0.40  10    0                                      R4137                            10    3                                 2    pATH2    Balb/C  M    Tubo 0.36  10    1                                      R4137                            10    7                                 3    pATH2    Balb/C  M    Tubo 0.36  25    5                                      R4137                            25    12                                4    pATH2    CD1     M    Tubo 0.36  37    10                                     R4137                            38    21                                5    pATH2    CD1     F    Tubo 0.29  20    6                                      R4137                            20    15                                6    pATH2    CD1     M    CTX  0.15  10    1                                      R4137                            10    10                                7    pATH2    CD1     M    CTX  0.15  21    6                                      R4137                            20    12                                ______________________________________                                    

A further experiment was carried out where mice were first injected witha lethal dose of cobra toxin and one hour later given either a placeboor the cholinergic decoyant. The animals which received the placebo alldied, whereas the decoy-treated animals were dramatically protected (90%survival). The results are shown in Table 2. It should be understoodthat the same receptor site is involved with all of BTX, CTX andd-tubocurarine as well as decamethonium and rabies-virus.

                  TABLE 2                                                         ______________________________________                                                       Time after toxin                                               Material injected                                                                            (minutes)   Survivors                                          ______________________________________                                        R4137 (decoyant)                                                                             30          6/10                                                              60          9/10                                                              90          6/7                                                pATH (placebo) 60          0/10                                               ______________________________________                                    

The fact that R4137 has been proven to be a decoyant against toxin isonly a case in point for the general claim of molecular decoyants astherapeutic agents. In the specific case of the AcChoR, R4137, orimproved versions of this molecular decoyant, can serve as an antidoteagainst cobra-like snake bites by specifically intercepting the α-toxinconstituent of these venoms. In addition, d-tubocurarine is routinelyused in surgery as a neuro-muscular blocking agent and a decoyant basedon R4137 could be extremely useful as its antidote. This utility is ofparticular importance as the use of ligand analogues as described in theprior art would be as detrimental as the blockers themselves. Moreover,it has recently been demonstrated that rabies-virus specifically bindsAcChoR and that its binding can be competed against d-tubocurarine orBTX (Lentz et al., Science, 215, 182-184 (1982)). Thus, a decoyant basedon R4137 will also provide therapeutic affects in the treatment ofrabies.

It should be understood that R4137 is but an intermediate tool which, byits genetic manipulation or chemical processing, would allow those ofordinary skill in the art to design even more efficient cholinergicdecoyants. Thus, the present invention is intended to include not onlythe specific 17 amino acid sequence of R4137, but variations andderivatives thereof which maintain, and preferably improve, itsfunctional or pharmacological characteristics. For example, modifiedpeptide sequences can be readily prepared and tested by routinetechniques for preferred toxin-binding characteristics so as to moreeffectively compete against the native receptor. Such modification mayinvolve substitution, deletion or insertion of amino acids or theirchemical modification. For example, longer lived decoyants may beobtained in this manner. As enzymatic degradation of the decoyants invivo may cause some decoyants to be relatively short-lived, one methodof preventing such degradation would be by making synthetic peptidescontaining d-amino acids. Alternatively, based on the fusion-proteinblueprint, organic molecules, i.e., not proteinaceous, can be designedso as to satisfy the physico-chemical requirements of a decoyant whichmust form a functional interface with the toxin.

It should further be understood that the decoyants of the presentinvention can be modified by extending the polypeptide or by addingspecific chemical moieties intended to aid in drug design or to permitthe decoyants to be used for additional utilities. One such modificationwould be to extend the polypeptide by moieties intended to affectsolubility, e.g., by the addition of a hydrophilic residue, such asserine, or a charged residue, such as glutamic acid. Furthermore, thedecoyant could be extended for the purpose of stabilization andpreservation of a desired conformation, such as by adding cysteineresidues for the formation of disulfide bridges.

Another reason to modify the decoyants would be to make the decoyantdetectable, even after administration. This might be done byradioiodination with a radioactive iodine isotope, directly, or byadding tyrosine for subsequent radioiodination. Such detectabledecoyants could be used to detect the presence and/or location ofspecified pathogenic agents or toxins. For example, detection of anaccumulation of R4137 in the area of a dog bite would indicate thepresence of rabies-virus. Thus, such detectable decoyants could be usedfor the selective detection and mapping of given foreign agents or fordiagnosis of the invasion of such an agent.

A further reason for modifying decoyants would be for acceleratedclearance of the conjugated foreign agent from the body. For example, adecoyant linked to an asialoglyco-moiety would be expected to be clearedby the liver. Thus, for example, a decoyant mimicking the receptor siteof an anti-cancer chemotherapeutic agent and containing such anasialoglyco-moiety, or any other moiety which would aid in itsclearance, could be used to inactivate and quickly remove excesschemotherapeutic agent after the therapy is completed in order to reduceside effects.

Proof of the effectiveness of R4137 as a decoyant against toxin in vivoestablishes the operability of the general concept of the presentinvention. Accordingly, it should be understood that the presentinvention comprehends not only decoyants based on the cholinergicbinding site but decoyants based on any endogenous receptor for aforeign agent which exerts an undesired effect only after binding tothat endogenous receptor. The first requirement of a decoyant inaccordance with the present invention is that it be a mimic of theendogenous receptor, i.e., it must functionally resemble the bindingsite, although it may differ physically. The term "functionallyresemble" means that the decoyant binds to the foreign agent in questionin a selective and specific manner and with reasonable affinity. For thepurpose of this invention, a receptor can be any ligand-binding moleculeas, for example, the ligand-binding site of a traditional cell-surfacereceptor, the substrate binding site of an enzyme, the ligand-bindingsite of gangliosides, etc.

It must be understood, however, that a decoyant in accordance with thepresent invention cannot be an immunoglobulin nor can it be derived fromimmunoglobulins. While it is true that an immunoglobulin directedagainst the "binding domain" of a-pathogen could generally function as adecoy, such is not intended to be included within the concept of thepresent invention.

A decoyant in accordance with the present invention should not besubstantially immunogenic. Reduction of size is a means of diminishingthe immunogenicity of a substance, but not all large molecules are asimmunogenic as some small molecules. To be classified as a decoyant inaccordance with the present invention, the substance must besubstantially non-immunogenic in the system of the host, regardless ofthe size of the substance, although the smallest possible size ispreferred. It is very important, however, that the decoyant not besufficiently immunogenic to elicit an autoimmune response against theendogenous receptor when administered in vivo. In the case of thecholinergic receptor, such an autoimmune response might cause a case ofmyasthenia gravis.

A decoyant in accordance with the present invention must comprise theessential elements of the binding site of a receptor and notsubstantially more. For the purpose of this invention, the "essentialelements" of a binding site are defined as those elements essential forthe decoyant activity, i.e., ligand recognition and binding. A receptorconsists of many residues, only a few of which are involved in ligandrecognition and binding. However, as discussed above, the decoyants ofthe present invention may be further modified for purposes of drugdesign. Thus, for example, the entire α-subunit of AcChoR would notqualify as a decoyant, being both immunogenic and also considerablylonger than necessary. The α-subunit does, however, contain thepotential information needed for the design and construction of adecoyant, i.e., sequence α-184-200. The fact that some additionalpeptide units may also be present, for example to improve the solubilityof the essential required sequence, would not remove the structure fromthe category of decoyant as long as it is still substantiallynon-immunogenic and it is still selective, specific and of reasonableaffinity. Addition of sugar molecules could be a modification with thesame effect. Thus, additions to the molecule for the purpose of drugdesign are not considered when determining whether the substancecontains substantially more than the elements of the endogenous receptorwhich are required for binding to the foreign agent in question.

As previously indicated, a decoyant must be selective, specific and ofreasonable affinity with respect to the agent for which it is designed.Thus, for example, mannose, a simple sugar, may interfere with infectionby bacteria that have type-I mannose specific pili; however, theselectivity of mannose is not sufficient and neither is its affinity.

A decoyant is a drug designed to intercept an invading foreign agenthaving an undesired effect. Such foreign agents may include toxins,poisons, bacteria, viruses, including retroviruses, etc. As long as theforeign agent exerts its pathogenic or toxic effect (or any other effectwhich is desired to be eliminated) only after binding to a receptor sitesomewhere in the host, a decoyant can be designed in accordance with thepresent invention to prevent such binding and thereby eliminate suchundesired effect. Once the procedures of the present invention areknown, as well as the fact that such receptor fractions containingligand binding sites will still competitively bind the ligands in vivo,those of ordinary skill in the art will understand that decoyantsderived from other receptors, designed to bind to other pathogenicagents and toxins, can be obtained using no more than routineexperimentation.

Among the pathogenic agent-receptor pairs for which decoyants canreadily be obtained in accordance with the present invention is theT-cell surface glycoprotein CD4 (T4), which is the cellular receptor forhuman immunodeficiency virus, type 1 (HIV-1), the first member of thefamily of viruses that cause acquired immunodeficiency syndrome (AIDS).The infection of the HIV virus starts through the binding of itsenvelope protein (gp120) to the T4 receptor (CD4) located on the T4lymphocytes. It has recently been confirmed that soluble, secreted formsof CD4 can be used to competitively bind HIV-1 and thus neutralize theinfectivity of HIV-1 (Smith et al, "Blocking of HIV-1 Infectivity by aSoluble, Secreted Form of the CD4 Antigen", Science, 238, 1704-1707(1987)). Intact CD4 would not be a decoyant in accordance with thepresent invention in view of its size. It does, however, contain theessence for a decoyant. The minimal binding domain of CD4 can beidentified using no more than routine experimentation by the meansdescribed herein for arriving at the minimal binding domain for thecholinergic receptor, e.g., by means of proteolysis and protein blottingfollowed by recombinant DNA procedures.

Another ligand-receptor pair particularly suited for the preparation ofdecoyants in accordance with the present invention isorganophosphate-acetylcholine esterase. Such a decoy would relieve someof the effects of nerve gas. Other examples are LSD and the serotoninreceptor and strychnine and the glycine receptor.

Table 3 shows additional ligand-receptor pairs for which decoyants inaccordance with the present invention can be designed using no more thanroutine experimentation:

                  TABLE 3                                                         ______________________________________                                        Ligand          Receptor                                                      ______________________________________                                        Calcium         28 kDa of bovine cerebellum and                                               kidney                                                                        Calmodulin                                                    Heparin         apoE and apoB of human plasma                                 Pili of Gonococcus                                                                            14 and 16 kDa proteins of CHO                                                 cells                                                         Virus                                                                         Retrovirus type 3                                                                             67 kDa glycoprotein of rodent                                                 lymphoid and neuronal cells                                   Sendai virus    Human erythrocyte glycophorin                                 Potato spindle tuber                                                                          Nuclear proteins                                              viroid                                                                        ______________________________________                                    

The decoyants of the present invention may be administered to an animal,including a human patient, in order to ameliorate the undesired effectsof the foreign agent for which it was designed. Such decoyants can beused not only for the treatment of humans, but also for the treatment ofother animals, including mammals, poultry, fish, etc. Furthermore,decoyants in accordance with the present invention could be designed forthe protection or treatment of plants. The specific effective dosagesfor the treatment of any given foreign agent can readily be empiricallydetermined by those of ordinary skill in the art without undueexperimentation. However, those skilled in the art will understand thatthe dosage of decoyant will depend to some extent on the amount offoreign agent in the system of the host. The ratio of decoyant toforeign agent molecules is preferably in the range of 1:1 to 1:10.Animal tests have shown that a large excess of decoyant is not necessaryfor effectiveness. Preferably, the amount of foreign agent in thebloodstream of the host will be monitored and the decoyant dosageadjusted accordingly during the course of treatment.

Compositions within the scope of the present invention includecompositions wherein the decoyant is present in an effective amount toachieve its intended purpose. Determination of the effective amounts iswithin the skill in the art.

In addition to the decoyants of the present invention, thepharmaceutical compositions may contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically. Preferably, the preparations, particularlythose which can be administered by injection, contain from about 0.1 to99 percent, and preferably from about 25 to 85 percent by weight, of theactive ingredient, together with the excipient.

Any conventional route of administration may be used for the decoyantsof the present invention. Although the preferred mode of administrationis by injection, e.g., intravenously, intradermally, intraperitoneally,etc, they may also be administered orally, by suppository or by anyother route.

Other non-conventional means of administration can be envisioned whichare also intended to be comprehended within the scope of the presentinvention. For example, while the present system for the expression ofthe active ingredient by bacteria involves the bacterial expressionvector pATH2, other bacterial expression systems exist which actuallysecrete the expressed protein into the medium. It could be contemplatedthat such a secreting expression system could be used to generate thedecoyant from within the host rather than producing it ex vivo andadministering it to the host. Obviously, the secreting system must becompatible with the host. The term "administration" as used in thepresent specification and claims is intended to include such in vivosecretion systems.

The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, dissolving, or lyophilizing processes. Suitableformulations for parenteral administration include aqueous solutions ofthe active compounds in water-soluble form. In addition, suspensions ofthe active compounds as appropriate oily injection suspensions may beadministered. Suitable lipophilic solvents or vehicles include fattyoils such as sesame oil, or synthetic fatty acid esters such as ethyloleate or triglycerides. Aqueous injection suspensions may containsubstances which increase the viscosity of the suspension such as sodiumcarboxymethyl cellulose, sorbitol, and/or dextran. optionally, thesuspension may also contain stabilizers. The decoyants of the presentinvention may also be administered in the form of liposomes,pharmaceutical compositions in which the active ingredient is containedeither dispersed or variously present in corpuscles.consisting ofaqueous concentric layers adherent to lipidic layers. The activeingredient may be present both in the aqueous layer and in the lipidiclayer, or, in any event, in the non-homogeneous system generally knownas a liposomic suspension.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without departing from the generic concept,and therefore such adaptations and modifications are intended to becomprehended within the meaning and range of equivalents of thedisclosed embodiments. It is to be understood that the phraseology andterminology employed herein is for the purpose of description and not oflimitation.

I claim:
 1. A method for the treatment of an organism which has beensubjected to a foreign agent which exerts an undesired effect only afterfirst binding to an endogenous receptor, said treatment being for theamelioration of said undesired effect, comprising:administering to saidorganism, in a quantity sufficient to ameliorate said undesired effectof said foreign agent, a molecular decoyant having a chemical structurewhich functionally resembles the ligand binding site of said endogenousreceptor for the foreign agent, said decoyant being a substance selectedfrom the group consisting of:(1) a substance which is a portion of anatural receptor for said foreign agent, said portion being (a)sufficiently small so as not to elicit an autoimmune response againstthe endogenous receptor when administered in vivo, and (b) notsubstantially larger than the smallest size needed to retain theelements of the binding site of said natural receptor which areessential for retaining the ability of said receptor to selectively andspecifically recognize and bind to said foreign agent with an affinitypermitting competition with binding of said foreign agent to saidendogenous receptor; (2) a substance having a chemical structure whichsubstantially corresponds to that of said substance of (1), synthesizedby chemical and/or recombinant DNA techniques and having the ability toselectively and specifically recognize and bind of the foreign agentwithout eliciting an autoimmune response against the endogenous receptorwhen administered in vivo; and (3) a substance having a chemicalstructure consisting essentially of the chemical structure of thesubstance of (1) or (2).
 2. A method in accordance with claim 1, whereinthe foreign agent is a snake venom toxin.
 3. A method in accordance withclaim 1, wherein the foreign agent is a rabies virus.
 4. A method inaccordance with claim 1, wherein the foreign agent is HIV virus.
 5. Amethod in accordance with claim 1, wherein said foreign agent is an HIVvirus and said endogenous receptor is the T4 cell antigen oflymphocytes.
 6. A method in accordance with claim 1, wherein saidmolecular decoyant is a substance of (1).
 7. A method in accordance withclaim 1, wherein said molecular decoyant is a peptide chain expressed bycells genetically engineered by recombinant DNA techniques.
 8. A methodin accordance with claim 1, wherein said molecular decoyant is asynthetic peptide.
 9. A method in accordance with claim 1, wherein saidorganism is an animal.
 10. A method in accordance with claim 9, whereinsaid animal is a human.
 11. A method in accordance with claim 1, whereinsaid decoyant is a substance of (1) or a substance having the samechemical structure as a substance of (1) synthesized by chemical and/orrecombinant DNA techniques.
 12. A method in accordance with claim 11,wherein said molecular decoyant is a peptide chain expressed by cellsgenetically engineered by recombinant DNA techniques.
 13. A method inaccordance with claim 11, wherein said molecular decoyant is a syntheticpeptide.
 14. A method in accordance with claim 1, wherein said substanceof (2) has the same chemical structure as the substance of (1)synthesized by chemical and/or recombinant DNA techniques.
 15. A methodfor the treatment of an organism which has been subjected to a foreignagent which exerts an undesired effect only after first binding to anendogenous receptor, said treatment being for the amelioration of saidundesired effect, comprising:administering to said organism, in aquantity sufficient to ameliorate said undesired effect of said foreignagent, a molecular decoyant having a chemical structure whichfunctionally resembles the ligand binding site of said endogenousreceptor for the foreign agent, said decoyant being a substance selectedfrom the group consisting of:(1) a substance which is a portion of anatural receptor for said foreign agent, said portion being (a)sufficiently small so as not to elicit an autoimmune response againstthe endogenous receptor when administered in vivo, and (b) notsubstantially larger than the smallest size needed to retain theelements of the binding site of said natural receptor which areessential for retaining the ability of said receptor to selectively andspecifically recognize and bind to said foreign agent with an affinitypermitting competition with binding of said foreign agent to saidendogenous receptor; (2) a substance having a chemical structure whichsubstantially corresponds to that of said substance of (1), synthesizedby chemical and/or recombinant DNA techniques and having the ability toselectively and specifically recognize and bind to the foreign receptorwithout eliciting an autoimmune response against the endogenous receptorwhen administered in vivo; and (3) a substance having a chemicalstructure consisting essentially of the chemical structure of thesubstance of (1) or (2); with the proviso that said endogenous receptoris not the CD4 receptor.
 16. A method for the treatment of an organismwhich has been subjected to a curarimimetic neurotoxin, which neurotoxinexerts an undesired effect only after first binding to a cholinergicreceptor, said treatment being for the amelioration of said undesiredeffect, comprising:administering to said organism, in a quantitysufficient to ameliorate said undesired effect of said curarimimeticneurotoxin, a molecular decoyant having a chemical structure whichfunctionally resembles the ligand binding site of said cholinergicreceptor, said decoyant being a substance selected from the groupconsisting of:(1) a substance which is a portion of a naturalcholinergic receptor, said portion being (a) sufficiently small so asnot to elicit an autoimmune response against the endogenous receptorwhen administered in vivo, and (b) not substantially larger than thesmallest size needed to retain the elements of the binding site of saidnatural receptor which are essential for retaining the ability of saidreceptor to selectively and specifically recognize and bind to saidcurarimimetic neurotoxin with an affinity permitting competition withbinding of said curarimimetic neurotoxin to said cholinergic receptor;(2) a substance having a chemical structure which substantiallycorresponds to that of said substance of (1), synthesized by chemicaland/or recombinant DNA techniques and having the ability to selectivelyand specifically recognize and bind to the curarimimetic neurotoxinwithout eliciting an autoimmune response against the endogenous receptorwhen administered in vivo; and (3) a substance having a chemicalstructure consisting essentially of the chemical structure of substance(1) or (2).
 17. A method in accordance with claim 16, wherein saidforeign agent is α-bungarotoxin, cobratoxin, d-tubocurarine,decamethonium, or rabies-virus, and said endogenous receptor is thenicotinic acetylcholine receptor.
 18. A method in accordance with claim16, wherein said molecular decoyant includes the amino acid sequenceTrp-Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr-Pro-Tyr-Leu-Asp,which molecular decoyant has the ability of selectively and specificallyrecognizing and binding to curarimimetic neurotoxin.
 19. A method inaccordance with claim 16, wherein said molecular decoyant includes anamino acid sequence substantially corresponding toTrp-Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr-Pro-Tyr-Leu-Asp,which molecular decoyant has the ability of selectively and specificallyrecognizing and binding to curarimimetic neurotoxin.
 20. A method forthe treatment of an organism which has been subjected to a foreign agentwhich exerts an undesired effect only after first binding to anendogenous receptor, said treatment being for the amelioration of saidundesired effect, comprising:administering to said organism, in aquantity sufficient to ameliorate said undesired effect of said foreignagent, a molecular decoyant which is a substance selected from the groupconsisting of:(1) a substance which is a portion of a natural receptorfor said foreign agent, said portion being not substantially larger thanthe smallest size needed to retain the elements of the binding site ofsaid natural receptor which are essential for retaining the ability ofsaid receptor to selectively and specifically recognize and bind to saidforeign agent with an affinity permitting competition with binding ofsaid foreign agent to said endogenous receptor; (2) a substance having achemical structure which substantially corresponds to that of saidsubstance of (1), synthesized by chemical and/or recombinant DNAtechniques and having the ability to selectively and specificallyrecognize and bind to the foreign agent with an affinity permittingcompetition with binding of said foreign agent to said endogenousreceptor; and (3) a substance having a chemical structure consistingessentially of the chemical structure of the substance of (1) or (2).21. A method in accordance with claim 20, wherein said decoyant is asubstance of (1) or a substance having the same chemical structure as asubstance of (1) synthesized by chemical and/or recombinant DNAtechniques.
 22. A method in accordance with claim 20, wherein saidsubstance of (2) has the same chemical structure as the substance of (1)synthesized by chemical and/or recombinant DNA techniques.
 23. A methodfor the treatment of an organism which has been subjected to acurarimimetic neurotoxin, which neurotoxin exerts an undesired effectonly after first binding to a cholinergic receptor, said treatment beingfor the amelioration of said undesired effect, comprising:administeringto said organism, in a quantity sufficient to ameliorate said undesiredeffect of said curarimimetic neurotoxin, a molecular decoyant which is asubstance selected from the group consisting of:(1) a substance which isa portion of a natural cholinergic receptor, said portion being notsubstantially larger than the smallest size needed to retain theelements of the binding site of said natural receptor which areessential for retaining the ability of said receptor to selectively andspecifically recognize and bind to said curarimimetic neurotoxin with anaffinity permitting competition with binding of said curarimimeticneurotoxin to said cholinergic receptor; (2) a substance having achemical structure which substantially corresponds to that of saidsubstance of (1), synthesized by chemical and/or recombinant DNAtechniques and having the ability to selectively and specificallyrecognize and bind to the curarimimetic neurotoxin with an affinitypermitting competition with binding of said curarimimetic neurotoxin tosaid cholinergic receptor; and (3) a substance having a chemicalstructure consisting essentially of the chemical structure of substance(1) or (2).
 24. A method in accordance with claim 23, wherein saiddecoyant is a substance of (1) or a substance having the same chemicalstructure as a substance of (1) synthesized by chemical and/orrecombinant DNA techniques.
 25. A method in accordance with claim 23,wherein said substance of (2) has the same chemical structure as thesubstance of (1) synthesized by chemical and/or recombinant DNAtechniques.
 26. A method in accordance with claim 23, wherein saidmolecular decoyant includes the amino acid sequenceTrp-Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr-Pro-Tyr-Leu-Asp,which molecular decoyant has the ability of selectively and specificallyrecognizing and binding to curarimimetic neurotoxin.